The helminth parasite Fasciola hepatica causes liver fluke disease or fascioliasis, thereby affecting the health of humans, as well as sheep, cattle and goats, among others mammalians. Fascioliasis causes losses in agriculture estimated at >US3.2billion per year. Fascioliasis is also a major zoonotic disease and it is estimated that 17 million people worldwide are infected. The flukicide triclabendazole is the most effective drug to control fascioliasis;however, the cost of treatment and the emergence of drug resistance in sheep infected with F. hepatica suggest a need to develop sustainable strategies, such as vaccination for the control of this disease. However, despite the long-standing research, a vaccine against Fasciola hepatica has not yet been developed. We have identified a novel F. hepatica antigen termed FhSAP2 that when delivered subcutaneously (SC) in Freund's complete adjuvant (FCA) is able to induce partial immunity (>60% reduction in parasite burden and significant reduction of liver damage) in rabbits and mice challenged with F. hepatica metacercariae. FhSAP2 is an 11.5kDa polypeptide belonging to the F. hepatica saposin-like / NK-lysin protein family. We also demonstrated that the immunity induced by the FhSAP2-FCA formulation is associated with high levels of IgG2a antibodies and high levels of IFN3, which are signatures of a Th1 immune response. These results led us to hypothesize that the protection induced by FhSAP2 is mediated by a mechanism linked to CD4+ Th1 cells, which is a new concept in the field of fascioliasis since F. hepatica, like most helminthes, is traditionally associated to Th2 immune responses. The goal of the present study is to study the modulation of T-helper 1, T- helper 2 responses and the protective effect against F. hepatica induced by FhSAP2 with these formulations in the mouse model of fascioliasis. To address this goal we propose to fulfill two specific aims in the 3-year funding period. In specific aim-1 we will study the protective effect of FhSAP2 when delivered SC in adjuvants containing immunostimulatory sequences. Specifically, we will ascertain whether the prime-boost strategy of naove C57BL/6 (H-2b) mice with FhSAP2 in MontanideTM ISM 1312 and ISA 70M or trapped into ISCOMs co- administered with IL-12 or CpG-ODN could mimic or enhance the protection levels obtained with FhSAP2 in CFA. This will lead to an optimization of the vaccine formulation for future studies. In the specific aim-2 we will study how the vaccine formulations used in the aim-1 modulate the T helper 1(Th1)-T helper 2 (Th2) responses. We will look at the Th1/Th2 cytokine profile, the levels of antibody subclasses as well as the activation status of B- and T-cells, macrophages (MX) and dendritic cells (DCs) elicited in naove mice and these parameters will be correlated with the levels of protection obtained in specific aim-1. The outcome will allow us to test our hypothesis of the protective role of Th1 responses in fascioliasis. If the proposed aims are achieved, we not only would have in hand an optimized vaccine formulation to be assayed in ruminants but we will also put forward a new concept of how to induce protection against F. hepatica, which will open the door to further studies to elucidate the immune mechanisms that make possible the Th1 immune responses which are effective against a multicellular fluke like F. hepatica. Moreover, these studies might serve as the basis for the application of this concept in the study of other neglected diseases caused by human liver fluke pathogens. The main objective of PI with this SC1 application is to make a transition to a non-SCORE support mechanism and in doing so establish herself as an independent competitive investigator. PUBLIC HEALTH RELEVANCE: Fasioliasis is a major disease of ruminants and more recently humans. We have demonstrated that an FhSAP2 antigen induces significant protection in a mouse and rabbit model of fascioliasis. We propose to optimize the vaccine formulation and improve the efficacy of the vaccine. In doing so in the future we will be able to evaluate the optimized vaccine in ruminants.